Several strategies of anti HIV intracellular immunization have been attempted over the last decade. These include the expression of either HIV-1 trans dominant proteins (i.e. Rev, Tat) (Fox et al., 1995; Fraiser et al, 1998; Hamm et al., 1999; Liu et al., 1994; Mautino et al., 2001; Plavec et al., 1997; Rossi et al., 1997), HIV-1 antisense sequences (Chadwick and Lever, 2000; Lavigne and Thierry, 1997; Shahabuddin and Khan, 2000), ribozymes targeting HIV-1 sequences (for a review, see Rossi, 2000), or intrabodies against HIV-1 proteins (Mhashilkar et al., 1995; Mhashilkar et al., 1999). An HIV-1 nef allele (F12nef) whose expression in trans (D'Aloja et al., 1998), or in cis (Olivetta et al., 2000) potently inhibits the HIV-1 replication was recently isolated and characterized . This anti HIV-1 phenotype strictly depends on the presence of three rare amino acid substitutions (i.e. G140E, V153L, and E177G) (D'Aloja et al., 2001). F12Nef acts against HIV-1 in both the processing of p55Gag polyprotein (Fackler et al., 2001), and the viral assembly/release (Olivetta et al., 2000). The consequence is that, as best described in the in cis expression system, the infecting HIV-1 becomes totally unable to release even non infectious viral particles (Olivetta et al., 2000). F12Nef is defective for a number of functions described for wild type Nef. In particular, it does not down regulate neither CD4 (D'Aloja et al., 1998), nor Class I major histocompatibility complex (MHC) membrane expression (M. Doria, personal communication), it does not activate the Nef associated kinase (NAK/PAK) (D'Aloja et al., 2001; Nunn and Marsh, 1996; Renkema et al., 1999; Sawai et al., 1994), and fails to interact with the V1H subunit of the V-ATPase (D'Aloja et al., 2001; Lu et al., 1998). On the other hand, F12Nef maintains the ability to dispose at the cell margin, as well as to interact with the CD4 intracytoplasmic tail, being both functions required for its antiviral phenotype (Olivetta et al., 2000).
However, the success of a gene engineering protocol relies on the sustained expression of both the transgene of interest and the gene selection marker. Frequently, this was attempted by inserting them under the control of different promoters. Unfortunately, this strategy frequently led to an unbalanced activity of the two promoters, due to the promoter interference. Considering that transduced cells are generally selected on the basis of the marker expression, unsatisfactory levels of the transgene expression could be often encountered.
The present invention seeks to overcome this problem and provide a means to selected cells with have been transduced with the transgene of interest and thus to provide a more efficient way to treat HIV infection.